Minimally Interventional Study on Prevalence of Emerging ESR1 Mutations in Liquid Biopsy in Three Cohorts of Patients With Breast Cancer in Comparison With Patient's Baseline ESR1 Mutation Status as Defined by Tissue Profiling.

This is a prospective minimally interventional biomarker cohort study.

Biomarkers:

• GS Focus Liquid Breast (liquid biopsy sequencing test, covering mutations in ESR1, PIK3CA, AKT1, PTEN, ERBB2, BRCA1, BRCA2, PALB2);
• GS Focus Breast (tissue sequencing assay performed on basal tissue biopsy from the primary tumour or metastatic lesion not exposed to any systemic therapy, covering the same gene panel);
• ESR1 mutation by πCode (PlexBioTM), which covers hotspot variants (K303R, E380Q, S463P, P535H, L536P, L536H, L536Q, L536R, Y537S, Y537N, Y537C and D538G), with analytical sensitivity ranging from 0.1% to 0.5%, depending on the variant;
• PTEN analysis by IHC (VENTANA SP218). Biomarker assessment will be carried out by NGS methodology in both liquid biopsy and baseline tissue biopsy. Both strategies consider a custom panel covering PIK3CA, AKT1, PTEN, ESR1, BRCA1, BRCA2, PALB2, ERBB2 genes.

NGS panels were internally validated and presented sensitivity, specificity and accuracy of 100,00% with a limit of detection of 0,5% VAF for liquid biopsy panel and sensitivity, specificity and accuracy of 100,00% with a limit of detection of 5% VAF for tissue panel.

Cohort 3 will be recruited in two phases. The pilot phase will include 30 patients to assess the complexity of recruitment, sample logistics and laboratory operations using the new πCode liquid biopsy assay. If approved by the investigators and the sponsor, recruitment will continue with a further 70 patients for full-scale implementation.

For the detection of ESR1 mutations in Cohort 3, πCode microdisc technology (PlexBio™) will be used with the IntelliPlex™ ESR1 Mutation cfDNA Kit. This assay covers hotspot mutations in ESR1 in exons 4, 5, 7 and 8 (12 variants), with a DNA input of 10 ng and a detection limit ranging from 0.1% to 0.5%.

In Cohort 3, an initial liquid biopsy NGS assay (GS Focus Liquid) will be collected 6 months after the start of treatment, followed by ctDNA analyses using GS Focus Liquid and the πCode assay every 3 months for each patient, provided there is no evidence of clinical/radiological progression or the emergence of an ESR1 mutation. In parallel, if archived paraffin blocks are available, tissue NGS (GS Focus) and PTEN IHC (see below) will be performed to assess alterations present in primary tissue or metastatic lesions prior to exposure to AI plus CDK4/6i.

PTEN IHC will be analyzed in samples from prospective cohorts 1, 2, and 3 with valid NGS results from tissue and residual paraffin blocks. For PTEN staining by IHC, the VENTANA SP218 antibody will be used, which binds to the PTEN protein in FFPE sample sections (OptiView DAB IHC detection kit) run on the Roche-Ventana Benchmark Ultra platform, according to the manufacturer's instructions.

Inclusion criteria

Cohort 1:

• BC patients, male and female,
• 18 years old and older,
• Pre or post menopausal,
• With HR+ (ER and/or PR positive) and Her-2 negative (confirmed centrally),
• Locally advanced irresectable and/or metastatic disease
• Confirmation of HR and Her-2 status may be performed in the primary tumor or in the metastatic lesion (patients with discordant results may be included),
• Patients must be candidates to CDK4/6i therapy in combination with endocrine therapy in the first line setting (with or without ovarian suppression)
• Patients may have received one previous line of chemotherapy in the metastatic setting, but no endocrine therapy in the metastatic setting is allowed,
• Patients may have received chemotherapy in the neo/adjuvant setting,
• Patients may have received endocrine therapy (with or without ovarian suppression) in the neo/adjuvant setting,
• Patients may have received a CDK4/6i in the adjuvant setting, provided they are still candidates for CDK4/6i therapy in the metastatic setting,
• Patients must be able to undergo a liquid biopsy procedure before starting their first line treatment,
• All patients must fill and sign an informed consent form.

Cohort 2:

• BC patients,
• Male and female,
• 18 years old and older,
• pre or post menopausal,
• With HR+ (ER and/or PR positive) and Her-2 negative (confirmed centrally),
• Locally advanced irresectable and/or metastatic disease who have progressed on a CDK4/6i in combination with endocrine therapy (with or without ovarian suppression) in the first or second line setting,
• All other non-conflicting inclusion criteria for cohort 1 apply.

Cohort 3:

• BC Patients,
• Male and female,
• 18 years or older,
• pre- or post-menopausal,
• With HR+ (ER and/or PR positive), HER2-negative (centrally confirmed) locally advanced unresectable and/or metastatic disease,
• Confirmation of HR and HER2 status may be performed on the primary tumour or the metastatic lesion (patients with discordant results may be included),
• Patients must be on first-line hormonal therapy with an aromatase inhibitor plus a CDK4/6 inhibitor (with or without ovarian suppression) for at least 6 months,
• No clinical or radiological evidence of progressive disease,
• Patients may have received a prior line of chemotherapy in the metastatic setting; however, prior endocrine therapy in the metastatic setting is not permitted,
• Patients may have received chemotherapy in the neoadjuvant/adjuvant setting,
• Patients may have received endocrine therapy (with or without ovarian suppression) in the neoadjuvant/adjuvant setting,
• Patients may have received a CDK4/6 inhibitor in the adjuvant setting, provided they are still eligible for CDK4/6 inhibitor therapy in the metastatic setting,
• Patients must be able to undergo a liquid biopsy procedure before starting their first-line treatment,
• All patients must read and sign an Informed Consent Form.

Exclusion criteria

• Patients WITHOUT HR+ (ER- and/or PR-positive)/HER2-negative disease,
• Patients without radiological and/or pathological confirmation of locally unresectable and/or metastatic breast cancer,
• Patients who are NOT candidates for further systemic treatment following a diagnosis of metastatic disease or disease progression,
• Patients who have already started a CDK4/6 inhibitor in combination with endocrine therapy (with or without ovarian suppression) for first-line metastatic disease (for Cohort 1);
• Patients who have already started a new line of treatment for metastatic disease following progression on a CDK4/6 inhibitor in combination with endocrine therapy (with or without ovarian suppression) (for Cohort 2);
• Patients with any suspicion of clinical or radiological disease progression at the time of the first liquid biopsy collection (for Cohort 3),
• Patients who are NOT able to undergo a liquid biopsy procedure,
• Patients who are NOT able to provide informed consent.